TCF3 Dominant Negative Variant Causes an Early Block in B-Lymphopoiesis and Agammaglobulinemia

ABSTRACT Interferon (IFN) mediates its antiviral effects by inducing a number of responsive genes, including the double-stranded RNA (dsRNA)-dependent protein kinase, PKR. Here we report that inducible overexpression of functional PKR in murine fibroblasts sensitized cells to apoptosis induced by influenza virus, while in contrast, cells expressing a dominant-negative variant of PKR were completely resistant. We determined that the mechanism of influenza virus-induced apoptosis involved death signaling through FADD/caspase-8 activation, while other viruses such as vesicular stomatitis virus (VSV) and Sindbis virus (SNV) did not significantly provoke PKR-mediated apoptosis but did induce cytolysis of fibroblasts via activation of caspase-9. Significantly, treatment with IFN-α/β greatly sensitized the fibroblasts to FADD-dependent apoptosis in response to dsRNA treatment or influenza virus infection but completely protected the cells against VSV and SNV replication in the absence of any cellular destruction. The mechanism by which IFN increases the cells' susceptibility to lysis by dsRNA or certain virus infection is by priming cells to FADD-dependent apoptosis, possibly by regulating the activity of the death-induced signaling complex (DISC). Conversely, IFN is also able to prevent the replication of viruses such as VSV that avoid triggering FADD-mediated DISC activity, by noncytopathic mechanisms, thus preventing destruction of the cell.

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Regulation of CCK-induced amylase release by PKC-δ in rat pancreatic acinar cells

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00111.2004 ◽

2004 ◽

Vol 287 (4) ◽

pp. G764-G771 ◽

Cited By ~ 40

Author(s):

Chenwei Li ◽

Xuequn Chen ◽

John A. Williams

Keyword(s):

Acinar Cells ◽

Adenoviral Vectors ◽

Dominant Negative ◽

Pancreatic Acinar Cells ◽

Amylase Secretion ◽

Wild Type ◽

Amylase Release ◽

Pkc Isoforms ◽

Chemical Inhibitors ◽

Dominant Negative Variant

PKC is known to be activated by pancreatic secretagogues such as CCK and carbachol and to participate along with calcium in amylase release. Four PKC isoforms, α, δ, ε, and ζ, have been identified in acinar cells, but which isoforms participate in amylase release are unknown. To identify the responsible isoforms, we used translocation assays, chemical inhibitors, and overexpression of individual isoforms and their dominant-negative variants by means of adenoviral vectors. CCK stimulation caused translocation of PKC-α, -δ, and -ε, but not -ζ from soluble to membrane fraction. CCK-induced amylase release was inhibited ∼30% by GF109203X, a broad spectrum PKC inhibitor, and by rottlerin, a PKC-δ inhibitor, but not by Gö6976, a PKC-α inhibitor, at concentrations from 1 to 5 μM. Neither overexpression of wild-type or dominant-negative PKC-α affected CCK-induced amylase release. Overexpression of PKC-δ and -ε enhanced amylase release, whereas only dominant-negative PKC-δ inhibited amylase release by 25%. PKC-δ overexpression increased amylase release at all concentrations of CCK, but dominant-negative PKC-δ only inhibited the maximal concentration; both similarly affected carbachol and JMV-180-induced amylase release. Overexpression of both PKC-δ and its dominant-negative variant affected the late but not the early phase of amylase release. GF109203X totally blocked the enhancement of amylase release by PKC-δ but had no further effect in the presence of dominant-negative PKC-δ. These results indicate that PKC-δ is the PKC isoform involved with amylase secretion.

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The Yeast AAA+ Chaperone Hsp104 Is Part of a Network That Links the Actin Cytoskeleton with the Inheritance of Damaged Proteins

Molecular and Cellular Biology ◽

10.1128/mcb.00201-09 ◽

2009 ◽

Vol 29 (13) ◽

pp. 3738-3745 ◽

Cited By ~ 57

Author(s):

Peter Tessarz ◽

Michael Schwarz ◽

Axel Mogk ◽

Bernd Bukau

Keyword(s):

Actin Cytoskeleton ◽

Dominant Negative ◽

Genetic Evidence ◽

Asymmetric Distribution ◽

Dominant Negative Variant ◽

Substrate Proteins

ABSTRACT The yeast AAA+ chaperone Hsp104 is essential for the development of thermotolerance and for the inheritance of prions. Recently, Hsp104, together with the actin cytoskeleton, has been implicated in the asymmetric distribution of carbonylated proteins. Here, we investigated the interplay between Hsp104 and actin by using a dominant-negative variant of Hsp104 (HAP/ClpP) that degrades substrate proteins instead of remodeling them. Coexpression of HAP/ClpP causes defects in morphology and the actin cytoskeleton. Taking a candidate approach, we identified Spa2, a member of the polarisome complex, as an Hsp104 substrate. Furthermore, we provided genetic evidence that links Spa2 and Hsp104 to Hof1, a member of the cytokinesis machinery. Spa2 and Hof1 knockout cells are affected in the asymmetric distribution of damaged proteins, suggesting that Hsp104, Spa2, and Hof1 are members of a network controlling the inheritance of carbonylated proteins.

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Oxysterol Binding Protein–related Protein 9 (ORP9) Is a Cholesterol Transfer Protein That Regulates Golgi Structure and Function

Molecular Biology of the Cell ◽

10.1091/mbc.e08-09-0905 ◽

2009 ◽

Vol 20 (5) ◽

pp. 1388-1399 ◽

Cited By ~ 117

Author(s):

Mike Ngo ◽

Neale D. Ridgway

Keyword(s):

Secretory Pathway ◽

Protein Transport ◽

Binding Protein ◽

Dominant Negative ◽

Ph Domain ◽

Oxysterol Binding Protein ◽

Large Gene ◽

Dominant Negative Variant

Oxysterol-binding protein (OSBP) and OSBP-related proteins (ORPs) constitute a large gene family that differentially localize to organellar membranes, reflecting a functional role in sterol signaling and/or transport. OSBP partitions between the endoplasmic reticulum (ER) and Golgi apparatus where it imparts sterol-dependent regulation of ceramide transport and sphingomyelin synthesis. ORP9L also is localized to the ER–Golgi, but its role in secretion and lipid transport is unknown. Here we demonstrate that ORP9L partitioning between the trans-Golgi/trans-Golgi network (TGN), and the ER is mediated by a phosphatidylinositol 4-phosphate (PI-4P)-specific PH domain and VAMP-associated protein (VAP), respectively. In vitro, both OSBP and ORP9L mediated PI-4P–dependent cholesterol transport between liposomes, suggesting their primary in vivo function is sterol transfer between the Golgi and ER. Depletion of ORP9L by RNAi caused Golgi fragmentation, inhibition of vesicular somatitus virus glycoprotein transport from the ER and accumulation of cholesterol in endosomes/lysosomes. Complete cessation of protein transport and cell growth inhibition was achieved by inducible overexpression of ORP9S, a dominant negative variant lacking the PH domain. We conclude that ORP9 maintains the integrity of the early secretory pathway by mediating transport of sterols between the ER and trans-Golgi/TGN.

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Glioblastoma invasion factor ODZ1 is induced by microenvironmental signals through activation of a Stat3-dependent transcriptional pathway

Scientific Reports ◽

10.1038/s41598-021-95753-6 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Veronica Vidal ◽

Olga Gutierrez ◽

Ana Talamillo ◽

Carlos Velasquez ◽

Jose L. Fernandez-Luna

Keyword(s):

Extracellular Matrix ◽

Matrix Protein ◽

Transmembrane Protein ◽

Gene Promoter ◽

Dominant Negative ◽

Surrounding Tissue ◽

Extracellular Matrix Protein ◽

Luciferase Reporter ◽

Dominant Negative Variant ◽

Transcriptional Pathway

AbstractWe have previously shown that the transmembrane protein ODZ1 serves for glioblastoma (GBM) cells to invade the surrounding tissue through activation of RhoA/ROCK pathway. However, the transcriptional machinery used by GBM cells to regulate the expression of ODZ1 is unknown. Here we show that interaction with tumor microenvironment elements, mainly activated monocytes through IL-6 secretion, and the extracellular matrix protein fibronectin, induces the Stat3 transcriptional pathway and upregulates ODZ1 which results in GBM cell migration. This signaling route is abrogated by blocking the IL-6 receptor, inhibiting Jak kinases or knocking down Stat3. Furthermore, we have identified a Stat3 responsive element in the ODZ1 gene promoter, about 1 kb from the transcription start site. Luciferase-reporter assays confirmed that the promoter responds to the presence of monocytic cells and this activation is greatly reduced when the Stat3 site is mutated or following treatment with a neutralizing anti-IL-6 receptor antibody or transfecting GBM cells with a dominant negative variant of Stat3. Overall, we show that monocyte-secreted IL-6 and the extracellular matrix protein fibronectin activate the axis Stat3-ODZ1 and promote migration of GBM cells. This is the first described transcriptional mechanism used by tumor cells to promote the expression of the invasion factor ODZ1.

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A synthetic amino acid substitution of Tyr10 in Aβ peptide sequence yields a dominant negative variant in amyloidogenesis

Aging Cell ◽

10.1111/j.1474-9726.2012.00814.x ◽

2012 ◽

Vol 11 (3) ◽

pp. 530-541 ◽

Cited By ~ 6

Author(s):

Honoree Mazargui ◽

Christian Lévêque ◽

Dirk Bartnik ◽

Jacques Fantini ◽

Tiphany Gouget ◽

...

Keyword(s):

Amino Acid ◽

Amino Acid Substitution ◽

Dominant Negative ◽

Peptide Sequence ◽

Aβ Peptide ◽

Synthetic Amino Acid ◽

Dominant Negative Variant

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Cellular interpretation of multiple TGF-beta signals: intracellular antagonism between activin/BVg1 and BMP-2/4 signaling mediated by Smads

Development ◽

10.1242/dev.124.22.4467 ◽

1997 ◽

Vol 124 (22) ◽

pp. 4467-4480 ◽

Cited By ~ 12

Author(s):

A.F. Candia ◽

T. Watabe ◽

S.H. Hawley ◽

D. Onichtchouk ◽

Y. Zhang ◽

...

Keyword(s):

Cell Fate ◽

Bone Morphogenetic Proteins ◽

Dominant Negative ◽

Signaling Cascades ◽

Mesoderm Induction ◽

Tgf Beta ◽

Functional Involvement ◽

Dominant Negative Variant ◽

Transcriptional Complex ◽

Mesoderm Patterning

During early embryogenesis of Xenopus, dorsoventral polarity of the mesoderm is established by dorsalizing and ventralizing agents, which are presumably mediated by the activity of an activin/BVg1-like protein and Bone Morphogenetic Proteins (BMP), respectively. Interestingly, these two TGF-beta subfamilies are found in overlapping regions during mesoderm patterning. This raises the question of how the presumptive mesodermal cells recognize the multiple TGF-beta signals and differentially interpret this information to assign a particular cell fate. In this study, we have exploited the well characterized model of Xenopus mesoderm induction to determine the intracellular interactions between BMP-2/4 and activin/BVg1 signaling cascades. Using a constitutively active BMP-2/4 receptor that transduces BMP-2/4 signals in a ligand-independent fashion, we demonstrate that signals provided by activin/BVg1 and BMP modulate each other's activity and that this crosstalk occurs through intracellular mechanisms. In assays using BMP-2/4 and activin/BVg1-specific reporters, we determined that the specificity of BMP-2/4 and activin/BVg1 signaling is mediated by Smad1 and Smad2, respectively. These Smads should be considered as the mediators of the intracellular antagonism between BMP-2/4 and activin/BVg1 signaling possibly through sequestration of a limited pool of Smad4. Consistent with such a mechanism, Smad4 interacts functionally with both Smad1 and −2 to potentiate their signaling activities, and a dominant negative variant of Smad4 can inhibit both activin/BVg1 and BMP-2/4 mediated signaling Finally, we demonstrate that an activin/BVg1-dependent transcriptional complex contains both Smad2 and Smad4 and thereby provides a physical basis for the functional involvement of both Smads in TGF-beta-dependent transcriptional regulation. Thus, Smad4 plays a central role in synergistically activating activin/BVg1 and BMP-dependent transcription and functions as an intracellular sensor for TGF-beta-related signals.

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Oncogenic conversion of the thyroid hormone receptor by altered nuclear transport

Nuclear Receptor Signaling ◽

10.1621/nrs.04008 ◽

2006 ◽

Vol 4 (1) ◽

pp. nrs.04008 ◽

Cited By ~ 12

Author(s):

Ghislain M.C. Bonamy ◽

Lizabeth A. Allison

Keyword(s):

Thyroid Hormone ◽

Signaling Pathways ◽

Hormone Receptor ◽

Thyroid Hormone Receptor ◽

Direct Interaction ◽

Dominant Negative ◽

Multiple Cell ◽

Dominant Negative Activity ◽

Dominant Negative Variant ◽

Complex Signaling

Nuclear receptors (NRs) are transcription factors whose activity is modulated by ligand binding. These receptors are at the core of complex signaling pathways and act as integrators of many cellular signals. In the last decade our understanding of NRs has greatly evolved. In particular, regulation of NR subcellular dynamics has emerged as central to their activity. Research on the subcellular distribution of the thyroid hormone receptor (TR) has revealed new dimensions in the complexity of NR regulation, and points to the possibility that NR mislocalization plays a key role in oncogenesis. For many years, TR was thought to reside exclusively in the nucleus. It is now known that TR is a dynamic protein that shuttles between the nucleus and cytoplasm. TR is localized to the nucleus in a phosphorylated form, suggesting that compartment-specific phosphorylation mediates cross-talk between multiple cell signaling pathways. The oncoprotein v-ErbA, a viral-derived dominant negative variant of TR is actively exported to the cytoplasm by the CRM1 export receptor. Strikingly, the oncoprotein causes mislocalization of cellular TR and some of its coactivators by direct interaction. Here, we offer some perspectives on the role of subcellular trafficking in the oncogenic conversion of TR, and propose a new model for oncoprotein dominant negative activity.

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